Opioid and cannabinoid receptor function is modulated by multiple cellular mechanisms; both receptors are critical to a number of physiological processes in health as well as in central nervous system disorders including the development of tolerance, dependence and addiction to drugs of abuse. This application focuses on the modulation of receptor function by direct protein-protein interaction i.e. dimerization. We and others have previously shown that opioid receptors dimerize with each other as well as with other G protein-coupled receptors such as cannabinoid receptors. In order to explore the molecular consequences of heterodimerization in vivo and its relevance to analgesia and addiction, we have initiated studies to characterize the mechanisms that contribute to the regulation of these heteromers. During the previous funding period we have made considerable progress with the characterization of opioid receptor heteromers in endogenous tissue. For this, we generated heteromer-selective antibodies and using them demonstrated that the heteromer levels are significantly upregulated in select brain regions under distinct pathological states. We also showed that these heteromers exhibit distinct signaling. Relatively little is known about the mechanisms regulating opioid receptor heteromer abundance or the functional relevance of heteromer signaling. In this application studies to investigate the mechanisms of regulation of opioid and cannabinoid receptor heteromers and to characterize heteromer-specific signaling are proposed. Specific aims are: Aim 1, to explore mechanisms of regulation of G protein-coupled receptor heteromer levels, and Aim 2, to generate, characterize, and validate tools that disrupt heteromers and use them to study heteromer-specific signaling. We describe studies using a combination of state-of-the-art technologies as well as unique reagents to address an emerging concept of disease-state specific heteromer upregulation. This, combined with heteromer-mediated distinct signaling, makes the heteromer an ideal disease-specific drug target. Studies proposed here, in addition to providing fundamental information about heteromer biology, form a foundation for the identification of small drug-like molecules specifically targeting the heteromer. Hence, the studies in this application are novel, pioneering and clinically relevant, and are likely to help i the identification of novel targets and therapeutics for the treatment of a number of brain diseases including drug addiction. PUBLIC HEALTH RELEVANCE: The exposure of opioid or cannabinoid receptors to drugs of abuse (morphine or tetrahydrocannabinol, respectively) initiates a number of biological responses including analgesia and development of tolerance/ addiction. The objective of studies in this grant application is to explore novel mechanisms such as receptor- receptor interactions in order to gain basic information about the early events that modulate receptor function. Furthermore, this information forms a foundation for studies identifying novel therapeutic targets for the treatment of pain and drug addiction.